In a uniplex PCR reaction (hereinafter referred to as uniplex PCR), the reaction typically contains template DNA, two primers flanking a single amplification site, a thermostable DNA polymerase, dNTPs and buffer. The resulting amplification products, the amplicons, are usually the target DNA fragment(s). The occasional non-specific amplification products are usually removed by fine-tuning PCR conditions, such as the annealing temperature, magnesium concentration, or through more stringent design of the primers.
Multiplex PCR refers to amplifying a plurality of target DNA fragments simultaneously in the same tube or well. This involves placing more than one pair of primers together. In practical applications, usually from tens to tens of thousands of different kinds of target DNA fragments are expected to be amplified simultaneously in a single tube. Multiplex PCR provides extraordinary simplicity, throughput, and economic advantages over uniplex PCR when different DNA fragments are needed to be amplified together in a single tube. It is also frequently used when dealing with precious samples (e.g., clinical samples).
Multiplex PCR already finds broad applications in detection and clinical diagnosis of genes and microorganisms in humans, animals, crops and plants, in species authentication, and more recently, in sample and library preparation for next generation sequencing (including, but not limited to, de novo sequencing and targeted re-sequencing). In gene testing and diagnosis, multiplex PCR is used in assays of single nucleotide polymorphisms (SNPs), genotyping, copy number variation, epigenetics, gene expression, and mutation and hybridization arrays.
Multiplex PCR typically requires placing together a large number of primers at certain concentrations in the same volume. These primers accumulate to very high quantity, for example up to micrograms, when a few thousand primers are used. A difficulty of multiplex PCR is that these primers can anneal with each other, leading to the generation of enormous amounts of non-specific amplification products. These non-specific amplification products usually make the target fragments undetectable, or simply cancel out the production of the target fragments.
Current techniques for making multiplex PCR workable despite the problem of these large amounts of primers and resulting background ‘noise’ may include specifically designing primers within very narrow parameter ranges, and/or incorporating exotic markers (e.g., U.S. Pat. Nos. 8,586,310 and 8,673,560, describe modification of primers by replacing thymidines with uredines so that the non-specific products are predominantly formed from non-specific annealing of primers that have multiple uridines interspersed along their length and can be specifically targeted on this basis), the use of oligonucleotides for preventing, or reducing, primer-dimer formation (see, e.g., WO2015063154 A1), and the use of tagged, target-specific primers that are blocked in combination with universal primers and a strategy for specifically activating blocked primers (see, e.g., US20140329245). All of these techniques have substantial drawbacks and limitations.
For example, methods for avoiding or reducing the formation of artifacts, such as primer-dimers, during nucleic acid amplification that center around the primer design process and often utilize dedicated software packages (e.g., DNAsoftwares's Visual OMP, MultiPLX, ABI's Primer Express, etc.) to design primer pairs that are predicted to exhibit minimal interaction between the other primers in the pool during amplification. Through the use of such software, primers can be designed to be as target-specific or amplicon-specific as possible, and often are grouped into subsets to minimize primer-primer interactions, primer-dimer formation and superamplicons. Stringent design parameters, however, limit the number of amplicons that can be co-amplified simultaneously and in some cases may prevent the amplification of some amplicons altogether. Other current methods require the use of multiple PCR primer pools to segregate primers into non-overlapping pools to minimize or prevent primer artifacts during the amplification step. Other methods include the use of multiple primer pools or single plex reactions to enhance the overall yield of amplification product per reaction.
In a multiplex PCR reaction, each primer pair competes in the amplification reaction with additional primer pairs for a finite amount of dNTPs, polymerase and other reagents. Primers may be designed to be longer than in uniplex PCR (24-35 bases), with higher melting temperatures (65° C. or higher) and a GC content of 50-60%, maintaining a similar melting temperature across all primers, and avoiding complementary sequences and runs of three or more Gs or Cs at the 3′ end; the specificity of each pair of primers is first validated in uniplex PCR to ensure a single target fragment is amplified; the concentration of primer pairs are titrated, usually from 50 nM to 400 nM to ensure uniformity of the yield; primers are segregated into non-overlapping pools; optimal concentrations of dNTPS, magnesium and the thermostable DNA polymerase are determined, which are usually higher than in uniplex PCR; and salt concentration is also titrated to optimize the amplification specificity. With these parameters optimized, usually 2-5 target fragments are successfully simultaneously amplified. Through further computer-aided design of primers, methods of amplifying ˜20 target fragments simultaneously in the same tube have been seen in the market. Due to the difficulty of mitigating or eliminating the non-specific products, only tens of targets are currently amplified in routine diagnostic practices. These methods require careful design of primers and PCR conditions, and fail to be used as standards for general diagnostic applications.
Numerous nested primers or similar strategies have been reported to extend the number of targets to be amplified in multiplex PCR. These methods may or may not always alleviate non-specific products generation. In addition, more primer layers, handling and cycling strategies are required in these methods. Therefore these methods find limited usage.
There is therefore a need for improved methods, compositions, systems, apparatuses and kits that allow for the selective amplification of multiple target nucleic acid molecules within a population of nucleic acid molecules while avoiding, or minimizing, the formation of artifacts (also referred to as non-specific amplification products), including primer dimers. There is also a need for improved methods, compositions, systems, apparatuses and kits that allow for the selective amplification of multiple target nucleic acid molecules from a single nucleic acid sample, such as genomic DNA and/or formalin-fixed paraffin embedded (FFPE) DNA while avoiding, or minimizing, the formation of artifacts. There is also a need in the art for improved methods, compositions, systems and kits that allow for the simultaneous amplification of thousands of target-specific nucleic acid molecules in a single reaction, which can be used in any applicable downstream assay or analysis. The methods, compositions, systems and kits described herein may address the problems and limitations discussed above.